S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

Neutrophilic Pleuritis Is a Severe Complication of Klebsiella pneumoniae Pneumonia in Old Mice.

The pathomechanisms underlying the frequently observed fatal outcome of Klebsiella pneumoniae pneumonia in elderly patients are understudied. In this study, we examined the early antibacterial immune response in young mice (age 2-3 mo) as compared with old mice (age 18-19 mo) postinfection with K. pneumoniae. Old mice exhibited significantly higher bacterial loads in lungs and bacteremia as early as 24 h postinfection compared with young mice, with neutrophilic pleuritis nearly exclusively developing in old but not young mice. Moreover, we observed heavily increased cytokine responses in lungs and pleural spaces along with increased mortality in old mice. Mechanistically, Nlrp3 inflammasome activation and caspase-1-dependent IL-1ß secretion contributed to the observed hyperinflammation, which decreased upon caspase-1 inhibitor treatment of K. pneumoniae-infected old mice. Irradiated old mice transplanted with the bone marrow of young mice did not show hyperinflammation or early bacteremia in response to K. pneumoniae. Collectively, the accentuated lung pathology observed in K. pneumoniae-infected old mice appears to be due to regulatory defects of the bone marrow but not the lung, while involving dysregulated activation of the Nlrp3/caspase-1/IL-1ß axis.

B Cells Are Not Involved in the Regulation of Adenoviral TGF-ß1- or Bleomycin-Induced Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is an irreversible, age-related diffuse parenchymal lung disease of poorly defined etiology. Many patients with IPF demonstrate distinctive lymphocytic interstitial infiltrations within remodeled lung tissue with uncertain pathogenetic relevance. Histopathological examination of explant lung tissue of patients with IPF revealed accentuated lymphoplasmacellular accumulations in close vicinity to, or even infiltrating, remodeled lung tissue. Similarly, we found significant accumulations of B cells interfused with T cells within remodeled lung tissue in two murine models of adenoviral TGF-ß1 or bleomycin (BLM)-induced lung fibrosis. Such B cell accumulations coincided with significantly increased lung collagen deposition, lung histopathology, and worsened lung function in wild-type (WT) mice. Surprisingly, B cell-deficient µMT knockout mice exhibited similar lung tissue remodeling and worsened lung function upon either AdTGF-ß1 or BLM as for WT mice. Comparative transcriptomic profiling of sorted B cells collected from lungs of AdTGF-ß1- and BLM-exposed WT mice identified a large set of commonly regulated genes, but with significant enrichment observed for Gene Ontology terms apparently not related to lung fibrogenesis. Collectively, although we observed B cell accumulations in lungs of IPF patients as well as two experimental models of lung fibrosis, comparative profiling of characteristic features of lung fibrosis between WT and B cell-deficient mice did not support a major involvement of B cells in lung fibrogenesis in mice.

Ectopic expression of C-type lectin Mincle renders mice susceptible to staphylococcal pneumonia.

Staphylococcus aureus is a prevalent pathogen in pneumonia and harbors glycolipids which may serve as molecular patterns in Mincle (Macrophage inducible C-type lectin) dependent pathogen recognition. We examined the role of Mincle in lung defense against S. aureus in WT, Mincle KO and Mincle transgenic (tg) mice. Two glycolipids, glucosyl-diacylglycerol (Glc-DAG) and diglucosyl-diacylglycerol (Glc2-DAG) were purified, of which only Glc-DAG triggered Mincle reporter cell activation and professional phagocyte responses. Proteomic profiling revealed that Glc2-DAG blocked Glc-DAG-induced cytokine responses, thereby acting as inhibitor of Glc-DAG/Mincle-signaling. WT mice responded to S. aureus with a similar lung pathology as Mincle KO mice, most likely due to Glc2-DAG-dependent inhibition of Glc-DAG/Mincle-signaling. In contrast, ectopic Mincle expression caused severe lung pathology in S. aureus-infected mice characterized by bacterial outgrowth and fatal pneumonia. Collectively, Glc2-DAG inhibits Glc-DAG/Mincle-dependent responses in WT mice, whereas sustained Mincle expression overrides Glc2-DAG-mediated inhibitory effects, conferring increased host susceptibility to S. aureus.

Role of matrix metalloprotease-2 and MMP-9 in experimental lung fibrosis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a diffuse parenchymal lung disease characterized by exuberant deposition of extracellular matrix (ECM) proteins in the lung interstitium, which contributes to substantial morbidity and mortality in IPF patients. Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases, many of which have been implicated in the regulation of ECM degradation in lung fibrosis. However, the roles of MMP-2 and -9 (also termed gelatinases A and B) have not yet been explored in lung fibrosis in detail. METHODS: AdTGF-ß1 was applied via orotracheal routes to the lungs of WT, MMP-2 KO, MMP-9 KO and MMP-2/-9 dKO mice on day 0 to induce lung fibrosis. Using hydroxyproline assay, FlexiVent based lung function measurement, histopathology, western blot and ELISA techniques, we analyzed MMP-2 and MMP-9 levels in BAL fluid and lung, collagen contents in lung and lung function in mice on day 14 and 21 post-treatment. RESULT: IPF lung homogenates exhibited significantly increased levels of MMP-2 and MMP-9, relative to disease controls. Enzymatically active MMP-2 and MMP-9 was increased in lungs of mice exposed to adenoviral TGF-ß1, suggesting a role for these metalloproteinases in lung fibrogenesis. However, we found that neither MMP-2 or MMP-9 nor combined MMP-2/-9 deletion had any effect on experimental lung fibrosis in mice. CONCLUSION: Together, our data strongly suggest that both gelatinases MMP-2 and MMP-9 play only a subordinate role in experimental lung fibrosis in mice.

Regulatory T Cells Limit Pneumococcus-Induced Exacerbation of Lung Fibrosis in Mice.

Patients with idiopathic pulmonary fibrosis (IPF) can experience life-threatening episodes of acute worsening of their disease, termed acute exacerbation of IPF, which may be caused by bacterial and/or viral infections. The potential for regulatory T cells (Tregs) to limit disease progression in bacterially triggered fibrosis exacerbation has not been explored so far. In the current study, we show that the number of Tregs was significantly increased in mice with established AdTGF-ß1-induced lung fibrosis and further increased in mice with pneumococcal infection-induced lung fibrosis exacerbation. Diphtheria toxin-induced depletion of Tregs significantly worsened infection-induced fibrosis exacerbation as determined by increased lung collagen deposition, lung histology, and elevated pulmonary Th1/Th2 cytokine levels. Conversely, IL-2 complex-induced Treg expansion in wild-type mice with established lung fibrosis completely inhibited pneumococcal infection-induced fibrosis exacerbation as efficaciously as antibiotic treatment while preserving lung antibacterial immunity in mice. Collectively, these findings demonstrate the efficacy of Tregs as "silencers," suppressing infection-induced exacerbation of lung fibrosis in mice, and their expansion may offer a novel adjunctive treatment to limit acute exacerbations in patients with IPF.

Overexpression of Macrophage-Inducible C-Type Lectin Mincle Aggravates Proinflammatory Responses to Streptococcus pneumoniae with Fatal Outcome in Mice.

Macrophage-inducible C-type lectin (Mincle)-dependent sensing of pathogens triggers proinflammatory immune responses in professional phagocytes that contribute to protecting the host against pathogen invasion. In this study, we examined whether overexpression of Mincle that is designed to improve early pathogen sensing by professional phagocytes would improve lung-protective immunity against Streptococcus pneumoniae in mice. Proteomic profiling of alveolar macrophages of Mincle transgenic (tg) mice stimulated with the Mincle-specific pneumococcal ligand glucosyl-diacylglycerol (Glc-DAG) revealed increased Nlrp3 inflammasome activation and downstream IL-1ß cytokine release that was not observed in Glc-DAG-stimulated Mincle knockout or Nlrp3 knockout macrophages. Along this line, Mincle tg mice also responded with a stronger Nlrp3 expression and early proinflammatory cytokine release after challenge with S. pneumoniae, ultimately leading to fatal pneumonia in the Mincle tg mice. Importantly, Nlrp3 inhibitor treatment of Mincle tg mice significantly mitigated the observed hyperinflammatory response to pneumococcal challenge. Together, we show that overexpression of the pattern recognition receptor Mincle triggers increased Glc-DAG-dependent Nlrp3 inflammasome activation in professional phagocytes leading to fatal pneumococcal pneumonia in mice that is amenable to Nlrp3 inhibitor treatment. These data show that ectopic expression of the Mincle receptor confers increased susceptibility rather than resistance to S. pneumoniae in mice, thus highlighting the importance of an inducible Mincle receptor expression in response to microbial challenge.

Loss of Serpina1 in Mice Leads to Altered Gene Expression in Inflammatory and Metabolic Pathways.

The SERPINA1 gene encodes alpha1-antitrypsin (AAT), an acute phase glycoprotein and serine protease inhibitor that is mainly (80-90%) produced in the liver. Point mutations in the SERPINA1 gene can lead to the misfolding, intracellular accumulation, and deficiency of circulating AAT protein, increasing the risk of developing chronic liver diseases or chronic obstructive pulmonary disease. Currently, siRNA technology can knock down the SERPINA1 gene and limit defective AAT production. How this latter affects other liver genes is unknown. Livers were taken from age- and sex-matched C57BL/6 wild-type (WT) and Serpina1 knockout mice (KO) aged from 8 to 14 weeks, all lacking the five serpin A1a-e paralogues. Total RNA was isolated and RNA sequencing, and transcriptome analysis was performed. The knockout of the Serpina1 gene in mice changed inflammatory, lipid metabolism, and cholesterol metabolism-related gene expression in the liver. Independent single-cell sequencing data of WT mice verified the involvement of Serpina1 in cholesterol metabolism. Our results from mice livers suggested that designing therapeutic strategies for the knockout of the SERPINA1 gene in humans must account for potential perturbations of key metabolic pathways and consequent mitigation of side effects.

Role of the COX2-PGE2 axis in S. pneumoniae-induced exacerbation of experimental fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) associated with high morbidity and mortality. Patients with ILD frequently develop an acute exacerbation of their disease, which may be triggered by viral and/or bacterial infections. Prostaglandin E2 (PGE2) is an eicosanoid released in a cyclooxygenase-2 (COX2)-dependent manner and is considered to contribute to regulation of lung fibrosis. However, its role in infection-induced exacerbation of lung fibrosis is poorly defined. We found significantly increased levels of PGE2 in lung tissue of patients with ILD. Increased levels of PGE2 were also found in lung tissue of mice with AdTGF-ß1-induced lung fibrosis and even more so in Streptococcus pneumoniae exacerbated lung fibrosis. Type II alveolar epithelial cells (AT II cells) and alveolar macrophages (AM) contributed to PGE2 release during exacerbating fibrosis. Application of parecoxib to inhibit PGE2 synthesis ameliorated lung fibrosis, whereas intratracheal application of PGE2 worsened lung fibrosis in mice. Both interventions had no effect on S. pneumoniae-exacerbated lung fibrosis. Together, we found that the COX2-PGE2 axis has dual roles in fibrosis that may offset each other: PGE2 helps resolve infection/attenuate inflammation in fibrosis exacerbation but accentuates TGF-ß/AT II cell-mediated fibrosis. These data support the efficacy of COX/PGE2 interventions in the setting of non-exacerbating lung fibrosis.

A comparison of curated gene sets versus transcriptomics-derived gene signatures for detecting pathway activation in immune cells.

BACKGROUND: Despite the significant contribution of transcriptomics to the fields of biological and biomedical research, interpreting long lists of significantly differentially expressed genes remains a challenging step in the analysis process. Gene set enrichment analysis is a standard approach for summarizing differentially expressed genes into pathways or other gene groupings. Here, we explore an alternative approach to utilizing gene sets from curated databases. We examine the method of deriving custom gene sets which may be relevant to a given experiment using reference data sets from previous transcriptomics studies. We call these data-derived gene sets, "gene signatures" for the biological process tested in the previous study. We focus on the feasibility of this approach in analyzing immune-related processes, which are complicated in their nature but play an important role in the medical research. RESULTS: We evaluate several statistical approaches to detecting the activity of a gene signature in a target data set. We compare the performance of the data-derived gene signature approach with comparable GO term gene sets across all of the statistical tests. A total of 61 differential expression comparisons generated from 26 transcriptome experiments were included in the analysis. These experiments covered eight immunological processes in eight types of leukocytes. The data-derived signatures were used to detect the presence of immunological processes in the test data with modest accuracy (AUC = 0.67). The performance for GO and literature based gene sets was worse (AUC = 0.59). Both approaches were plagued by poor specificity. CONCLUSIONS: When investigators seek to test specific hypotheses, the data-derived signature approach can perform as well, if not better than standard gene-set based approaches for immunological signatures. Furthermore, the data-derived signatures can be generated in the cases that well-defined gene sets are lacking from pathway databases and also offer the opportunity for defining signatures in a cell-type specific manner. However, neither the data-derived signatures nor standard gene-sets can be demonstrated to reliably provide negative predictions for negative cases. We conclude that the data-derived signature approach is a useful and sometimes necessary tool, but analysts should be weary of false positives.

Basophils enhance immunological memory responses.

The cellular basis of immunological memory remains a controversial issue. Here we show that basophils bound large amounts of intact antigens on their surface and were the main source of interleukins 6 and 4 in the spleen and bone marrow after restimulation with a soluble antigen. Depletion of basophils resulted in a much lower humoral memory response and greater susceptibility of immunized mice to sepsis induced by Streptococcus pneumoniae. Adoptive transfer of antigen-reactive basophils significantly increased specific antibody production, and activated basophils, together with CD4(+) T cells, profoundly enhanced B cell proliferation and immunoglobulin production. These basophil-dependent effects on B cells required interleukins 6 and 4 and increased the capacity of CD4(+) T cells to provide B cell help. Thus, basophils are important contributors to humoral memory immune responses.

Surfactant dysfunction and alveolar collapse are linked with fibrotic septal wall remodeling in the TGF-ß1-induced mouse model of pulmonary fibrosis.

In human idiopathic pulmonary fibrosis (IPF), collapse of distal airspaces occurs in areas of the lung not (yet) remodeled. Mice lungs overexpressing transforming growth factor-ß1 (TGF-ß1) recapitulate this abnormality: surfactant dysfunction results in alveolar collapse preceding fibrosis and loss of alveolar epithelial type II (AE2) cells' apical membrane surface area. Here we examined whether surfactant dysfunction-related alveolar collapse due to TGF-ß1 overexpression is linked to septal wall remodeling and AE2 cell abnormalities. Three and 6 days after gene transfer of TGF-ß1, mice received either intratracheal surfactant (Surf-groups: Curosurf®, 100 mg/kg bodyweight) or 0.9% NaCl (Saline-groups). On days 7 (D7) and 14 (D14), lung mechanics were assessed followed by design-based stereology at light and electron microscopic level to quantify structures. Compared with Saline, Surf showed significantly improved tissue elastance, increased numbers of open alveoli, as well as reduced alveolar size heterogeneity on D7. Deterioration in lung mechanics was highly correlated to the loss of open alveoli. On D14, lung mechanics, number of open alveoli, and alveolar size heterogeneity remained significantly improved in the Surf-group. Volumes of extracellular matrix and collagen fibrils in septal walls were significantly reduced, whereas the apical membrane surface area of AE2 cells was increased in Surf compared with Saline. In remodeled tissue with collapsed alveoli, three-dimensional reconstruction of AE2 cells based on scanning electron microscopy array tomography revealed that AE2 cells were trapped without contact to airspaces in the TGF-ß1 mouse model. Similar observations were made in human IPF. Based on correlation analyses, the number of open alveoli and of alveolar size heterogeneity were highly linked with the loss of apical membrane surface area of AE2 cells and deposition of collagen fibrils in septal walls on D14. In conclusion, surfactant replacement therapy stabilizes alveoli and prevents extracellular matrix deposition in septal walls in the TGF-ß1 model.

The FMS-like tyrosine kinase-3 ligand/lung dendritic cell axis contributes to regulation of pulmonary fibrosis.

RATIONALE: Dendritic cells (DC) accumulate in the lungs of patients with idiopathic lung fibrosis, but their pathogenetic relevance is poorly defined. OBJECTIVES: To assess the role of the FMS-like tyrosine kinase-3 ligand (Flt3L)-lung dendritic cell axis in lung fibrosis. MEASUREMENTS AND MAIN RESULTS: We demonstrate in a model of adenoviral gene transfer of active TGF-ß1 that established lung fibrosis was accompanied by elevated serum Flt3L levels and subsequent accumulation of CD11bpos DC in the lungs of mice. Patients with idiopathic pulmonary fibrosis also demonstrated increased levels of Flt3L protein in serum and lung tissue and accumulation of lung DC in explant subpleural lung tissue specimen. Mice lacking Flt3L showed significantly reduced lung DC along with worsened lung fibrosis and reduced lung function relative to wild-type (WT) mice, which could be inhibited by administration of recombinant Flt3L. Moreover, therapeutic Flt3L increased numbers of CD11bpos DC and improved lung fibrosis in WT mice exposed to AdTGF-ß1. In this line, RNA-sequencing analysis of CD11bpos DC revealed significantly enriched differentially expressed genes within extracellular matrix degrading enzyme and matrix metalloprotease gene clusters. In contrast, the CD103pos DC subset did not appear to be involved in pulmonary fibrogenesis. CONCLUSIONS: We show that Flt3L protein and numbers of lung DC are upregulated in mice and humans during pulmonary fibrogenesis, and increased mobilisation of lung CD11bpos DC limits the severity of lung fibrosis in mice. The current study helps to inform the development of DC-based immunotherapy as a novel intervention against lung fibrosis in humans.

Colonization-induced protection against invasive pneumococcal disease in mice is independent of CD103 driven adaptive immune responses.

Nasopharyngeal colonization with Streptococcus pneumoniae (the pneumococcus) is known to mount protective adaptive immune responses in rodents and humans. However, the cellular response of the nasopharyngeal compartment to pneumococcal colonization and its importance for the ensuing adaptive immune response is only partially defined. Here we show that nasopharyngeal colonization with S. pneumoniae triggered substantial expansion of both integrin αE (CD103) positive dendritic cells (DC) and T lymphocytes in nasopharynx, nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) of WT mice. However, nasopharyngeal de-colonization and pneumococcus-specific antibody responses were similar between WT and CD103 KO mice or Batf3 KO mice. Also, naïve WT mice passively immunized with antiserum from previously colonized WT and CD103 KO mice were similarly protected against invasive pneumococcal disease (IPD). In summary, the data show that CD103 is dispensable for pneumococcal colonization-induced adaptive immune responses in mice.

Nasopharyngeal colonization with Streptococcus pneumoniae triggers dendritic cell dependent antibody responses against invasive disease in mice.

Nasopharyngeal colonization with Streptococcus pneumoniae (Spn) is an important precondition for the development of pneumococcal pneumonia. At the same time, nasopharyngeal colonization with Spn has been shown to mount adaptive immune responses against Spn in mice and humans. Cellular responses of the nasopharyngeal compartment, including the nasal-associated lymphoid tissue, to pneumococcal colonization and their importance for developing adaptive immune responses are poorly defined. We show that nasopharyngeal colonization with S. pneumoniae led to substantial expansion of dendritic cells (DCs) both in nasopharyngeal tissue and nasal-associated lymphoid tissue of mice. Depletion of DCs achieved by either diphtheria toxin (DT) treatment of chimeric zDC+/DTR mice, or by use of FMS-like tyrosine kinase 3 ligand (Flt3L) KO mice exhibiting congenitally reduced DC pool sizes, significantly diminished antibody responses after colonization with Spn, along with impaired protective immunity against invasive pneumococcal disease. Collectively, the data show that classical DCs contribute to pneumococcal colonization induced adaptive immune responses against invasive pneumococcal disease in two different mouse models. These data may be useful for future nasopharyngeal vaccination strategies against pneumococcal diseases in humans.

Optimising experimental research in respiratory diseases: an ERS statement.

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age, ex vivo and in vitro models, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia.

Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

Cathepsin G in Experimental Tuberculosis: Relevance for Antibacterial Protection and Potential for Immunotherapy.

Neutrophil serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), have been implicated in the protective response against infections, including experimental mycobacterial infections. The goal of this study was to explore the role of CG in immunocompetent mice challenged aerogenically with Mycobacterium tuberculosis. We used genetically CG- or CG/NE-deficient mice to define the importance of these neutrophil serine proteases for antibacterial protection, granulomatous response, and survival. In addition, we explored the effect of intratracheally delivered liposomally encapsulated CG/NE as a therapeutic approach early during M. tuberculosis infection. Our data show that the presence of CG or CG/NE prolongs survival in M. tuberculosis-infected mice. However, CG is not directly involved in antibacterial defenses, and exogenous intratracheal administration of CG combined with NE does not reduce bacterial loads in the lungs of M. tuberculosis-infected mice.

Surfactant dysfunction during overexpression of TGF-ß1 precedes profibrotic lung remodeling in vivo.

Transforming growth factor-ß1 (TGF-ß1) is involved in regulation of cellular proliferation, differentiation, and fibrogenesis, inducing myofibroblast migration and increasing extracellular matrix synthesis. Here, TGF-ß1 effects on pulmonary structure and function were analyzed. Adenovirus-mediated gene transfer of TGF-ß1 in mice lungs was performed and evaluated by design-based stereology, invasive pulmonary function testing, and detailed analyses of the surfactant system 1 and 2 wk after gene transfer. After 1 wk decreased static compliance was linked with a dramatic alveolar derecruitment without edema formation or increase in the volume of septal wall tissue or collagen fibrils. Abnormally high surface tension correlated with downregulation of surfactant proteins B and C. TTF-1 expression was reduced, and, using PLA (proximity ligand assay) technology, we found Smad3 and TTF-1 forming complexes in vivo, which are normally translocated into the nucleus of the alveolar epithelial type II cells (AE2C) but in the presence of TGF-ß1 remain in the cytoplasm. AE2C show altered morphology, resulting in loss of total apical surface area per lung and polarity. These changes of AE2C were progressive 2 wk after gene transfer and correlated with lung compliance. Although static lung compliance remained low, the volume of septal wall tissue and collagen fibrils increased 2 wk after gene transfer. In this animal model, the primary effect of TGF-ß1 signaling in the lung is downregulation of surfactant proteins, high surface tension, alveolar derecruitment, and mechanical stress, which precede fibrotic tissue remodeling and progressive loss of AE2C polarity. Initial TTF-1 dysfunction is potentially linked to downregulation of surfactant proteins.

Arginase 1 activity worsens lung-protective immunity against Streptococcus pneumoniae infection.

Type 2 helper cell (Th2) dominated chronic lung diseases such as asthma are characterized by an increased risk for bacterial lung infections. However, the underlying mechanisms are poorly defined. Arginase 1 (Arg1) has been suggested to play an important role in the pathophysiology of asthma, and is rapidly induced in lung macrophages by Th2 cytokines, thereby limiting macrophage-derived antimicrobial nitric oxide (NO) production. Here we examined the effect of Th2 cytokine induced upregulation or lung myeloid cell specific conditional knockdown of Arg1 on lung resistance against Streptococcus pneumoniae (Spn) in mice. Lung macrophages responded with a profound induction of Arg1 mRNA and protein to treatment with IL-13 both in vitro and in vivo. IL-13-induced Arg1 activity in the lungs of mice led to significantly attenuated lung-protective immunity against Spn, while conditional Arg1 knockdown had no effect on lung-protective immunity against Spn. Collectively, the data show that Th2 cytokine induced increased Arg1 activity worsens lung-protective immunity against Spn, and interventions to block Th2 cytokine induced lung Arg1 activity may thus be a novel immunomodulatory strategy to lower the risk of bacterial infections in asthmatic patients.